PROJECT SUMMARY/ABSTRACT Ovarian cancer (OC) is the most lethal gynecological cancer in developed countries and is the fifth leading cause of cancer-related death among women. Angiogenesis plays a crucial role in the progression of growth and metastasis of all solid tumors including OC. In addition to angiogenesis, tumor cells may directly form and line vascular channels that can anastamose with normal blood vessels, a process known as vasculogenic mimicry (VM). OC is one of the earliest carcinomas in which VM was described. VM has been shown to correlate with increased tumor aggressiveness, metastatic disease and decreased survival. Although the VEGF antibody bevacizumab has been approved by the FDA for use in combination with chemotherapy for the treatment of advanced OC, up until now therapeutic targeting of either VM alone, or angiogenesis and VM combination has been largely unexplored. Since both VM and angiogenesis play a critical role in supporting growth of many solid cancers including breast and OCs, we hypothesize that the combined inhibition of angiogenesis and VM will be a more potent means of inhibiting growth of such cancers in vivo. We have developed a unique ?targeted anti- vascular strategy? that combines inhibition of both VM and angiogenesis. Our strategy is based on the generation of a fusion protein where a cancer targeted antibody is C-terminally fused with a biological ?payload?, endostatin- P125A (huEndo-P125A containing an alanine substitution of proline 125 which significantly increases the anti- angiogenic potency of wild type endostatin). Anti-EGFR-huEndo-P125A is the first candidate within this strategy that we have been testing in Triple Negative Breast Cancer (TNBC) xenograft models, and for which we were awarded SBIR Phase I grant (1R43CA195882). Anti-EGFR-huEndo-P125A demonstrates the ability to inhibit both angiogenesis and VM by TNBC cells in vitro, and also demonstrated significant inhibition of metastasis in vivo in TNBC xenograft models. We now propose extension of this strategy to HER2-expressing OC using a newly synthesized fusion protein, ?-HER2-IgG1-huEndo-P125A. In prior experiments with a prototype ?HER2- IgG3-huEndo-P125A we have already observed profound anti-angiogenic and VM inhibition of normal endothelial cells and OC cells, respectively, and substantial inhibition of growth of OC xenografts in vivo. We now propose testing of an IgG1 based fusion, since human IgG3 MAbs have shorter half-lives, a long and proteolysis-labile hinge region, numerous allotypic polymorphisms and are inferior to IgG1 in mediating ADCC. The Effects of the currently proposed ?HER2-IgG1-huEndo-P125A fusion on angiogenesis and VM inhibition will be determined by in vitro Matrigel assays, while SKOV-3 as well as established patient-derived xenograft (PDX) models will be utilized to test effects on tumor progression and metastasis by the fusion alone, in combination with chemotherapy and/or combined with bevacizumab. These studies will provide preclinical data towards filing of an IND and human Phase I testing in advanced ovarian cancer.